The effects of spironolactone administration on the activities of adrenal 21-hydroxylases was examined in male cortisol-and corticosterone-producing animals. Decreases in the activities of the 21-hydroxylases after spironolactone treatment occur only in those animals which produce predominantly cortisol rather than corticosterone and which have a high activity of adrenal steroid 17 alpha-hydroxylase. The administration of spironolactone to cortisol-producing animals, namely the guinea pig and the dog, caused a 50 to 75% loss in the activities of adrenal 21-hydroxylases, with a concomitant decrease in the content of microsomal cytochrome P-450, microsomal heme, and in the activity of microsomal 17 alpha-hydroxylase. Spironolactone treatment was also found to decrease the content of adrenal mitochondrial cytochrome P-450 in male guinea pigs, but not in male dogs. In contrast to its effect in cortisol-producing animals, the administration of spironolactone caused an increase in the activities of the microsomal 21-hydroxylases in the adrenals of corticosterone-producing animals such as the rat and the rabbit. BIBLIOGRAPHIC REFERENCES: Menard, R.H., S.A. Latiff, and J.L. Purvis. Intratesticular localization of cytochrome P-450 and 17 alpha-hydroxylase in the rat testis. Endocrinology 97, 1587 (1975). Purvis, J.L., and R.H. Menard. Compartmentation of Microsomal Cytochrome P-450 and 17 alpha-hydroxylase Activity in the Rat Testis in Hormonal Regulation of Spermatogenesis (F.S. French, ed.), Plenum Press, N.Y. (1976).